The so-called in vitro fertilization and embryo transfer comprises fertilizing ova and sperm in vitro and then transplanting the developed embryos into a female body. Since the world's first case of a human birth was reported in England in 1978 by Edwards et.al., and along with recent progress in the developmental technology, this treatment has been rapidly and widely used in Japan, and it is now an indispensable treatment for sterility.
However, there are still few cases that lead to pregnancy by the method, and it can not be said that the method is completely established. This may be because of the lower fertility in sterile patients due to male factor, but the lower implantation rate of ova to be transplanted seems to be main cause ((Mori,Munehide et.al., Nippon sankahujin kagakukai zashi, v.45, p.397(1993); Cohen,J. et al., VIIIth World Congress on in vitro Fertilization and Alternate Assisted Reproduction Kyoto, Sep., 12-15(1993), World Collaborative Report(1991)).
In addition to the technical factors, a lowering in the quality of embryos during culture seems to be responsible for such lower implantation rates (Inoue, Masahito, Rinsho fujinka sanka, v.48, p.148(1994)). That is to say, ova of mammalia do not have substances that correspond to the albumin in the eggs of reptiles and birds, and therefore, the amounts of nutrient reserved in the mammalian ova are naturally low. Thus, in the early embryos of in vitro fertilization, nutrient factors must be taken up through the zona pellucida from the culture medium. However, chemically defined media such as Ham's F-10 medium, MEM (Minimum Essential Medium), Dulbecco's MEM and the like, which have been conventionally utilized in in vitro fertilization, were not originally composed for the purpose of in vitro fertilization, but they are the media used conventionally in tissue culture or their modified media, therefore it cannot be said that they are the optimal media for the early embryos regarding the nutrient composition.
Recently, HTF medium (Human Tubal Fluid Medium) has been developed as nutriologically suitable medium for human in vitro fertilization, with a composition approximating to the electrolyte of human oviduct fluid (Quinn,P. J. et al., Fertility and Sterility, v.44, p.493(1982)). The medium is commercially available and replaces Ham's F-10 medium that was used predominantly so far. However, because the HTF medium only contains electrolytes as the main components and glucose as an energy source, the HTF medium shows no improvement over the Ham's F-10 medium containing amino acids, as regards the nutrient composition. In fact, despite the use of this medium, the implantation rate can not be enhanced though any substantial improvement in the problem of the lowering in quality of embryos.
In order to make up for this disadvantage, a method has been utilized in which the embryos are fed by adding the female serum which has been inactivated by heat treatment to the medium. The serum contains growth factors and the like, in addition to proteins, carbohydrates, lipids, vitamins and minerals as five nutrients which are essential factors in animal cell culture. For reason, the serum is added during embryo culture.
However, it has been reported that such serum is not always needed in the in vitro fertilization-embryo transfer process (Menezo,Y. et al., Fertility and Sterility v.42, p.750(1984)) and that on the contrary the growth of embryos may be suppressed by the addition of serum (Mehita, et al.Biology of Reproduction v.43, p.600 (1990)). Also, the serum itself is troublesome to collect and there is a danger of contamination by viruses etc. Therefore, the serum is not suitable as an additive for the medium of an in vitro fertilized ova.
At present, the substance which is mostly noted as the embryo growth-suppressing factor is oxygen free radical. This is based on the idea that the growth of embryos is suppressed by oxidative stress due to more opportunities of direct contact with oxygen in vitro, compared with in vivo (Whitten,W., Advanced in the Biosciences v.6, p.129 (1971); Quinn, P. J. et al., Journal of Experimental Zoology, v.206, p.73 (1978)). On the basis of this idea, it has been tried to prevent such oxidative stress for enhancing the growth of embryos by adding superoxide dismutase (SOD), edetic acid (EDTA) and the like to a medium (Abramczuk, J. et al., Developmental Biology, v.61, p.378 (1977); Nonozaki, T. et al., Journal of Assisted Reproduction and Genetics, v.9, p.274 (1992)).
It has also been reported that co-cultures using the epithelial cells of the oviduct whose effective components are unknown are effective for the growth of embryos (Xu, K. P. et al., Journal of Reproduction and Fertility, v.94, p.33 (1992)) and that a growth factor such as an insulin-like growth factors directly stimulates the growth of embryos (Matui, Motozumi et al., Honyudoubutu ranshi gakkaishi, v.11, p.132 (19949).
However, an analysis result has also been reported in which such a co-culture is, at most, effective for the detoxification of a medium and there is no evidence available of feeding to the embryos (Bavister, B. D., Human Reproduction, v.7, p.1339 (1992)). In any event, any conventional media for in vitro fertilization and any methods for adding additives to them as described above, including the addition of superoxide dismutase, EDTA and the like, merely partially prevent the cessation of the growth in vitro. Furthermore, there are very inconvenient to handle because, in actual culture of embryos, the optimal media corresponding to the embryo's growth stages must be suitably selected and exchanged at every stages.
Accordingly, in this technical field, the development of a chemically defined medium which contains a nutrient composition suitable for treatment/pretreatment of sperm or ovum as well as for growing of early embryo in the in vitro fertilization-embryo transfer process, which can be applied to all the growth stages of early embryo, and which is safe and leads to no danger of possible contamination of toxic substances such as viruses has been demanded.